Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor

Although Toll-like receptors have been widely identified and functionally characterized in mammalian models and Drosophila, the immunological function of these receptors in other insects remains unclear. Here, we explored the relevant innate immune response of Tenebrio molitor (T. molitor) Toll-3 against Gram-negative bacteria, Gram-positive bacteria, and fungal infections. Our findings indicated that TmToll-3 expression was mainly induced by Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of young T. molitor larvae. Surprisingly, Escherichia coli systemic infection caused mortality after TmToll-3 knockdown via RNA interference (RNAi) injection, which was not observed in the control group. Further analyses indicated that in the absence of TmToll-3, the final effector of the Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated after E. coli challenge. Our results indicated that the expression of almost all AMP genes was suppressed in silenced individuals, whereas the expression of relevant genes was positively regulated after fungal injection. Therefore, this study revealed the immunological involvement of TmToll-3 in T. molitor in response to systematic infections.


Introduction
Toll and Toll-like receptors (TLRs) play a major role in the innate immunity of insects and mammals [1]. Toll, which was the first of these receptors to be discovered, was initially noted for its role in dorsal-ventral patterning in Drosophila embryos [2] and was later shown to participate in the immune response against fungi and Gram-positive bacteria in larvae and adults [3]. Much of what is known about Toll receptors in insects derives from studies on Drosophila melanogaster, and a total of nine Toll receptors (Toll, Toll-2 also known as 18-Wheeler, and Toll-3 to Toll-9) have thus far been identified in this model [4]. Except for the Toll-3 (also known as MstProx) and Toll-4 genes, which have no clear functional or phenotypic characteristics [5], most of the Drosophila Toll paralogs (Toll-1, -2, -5, -6, -7, -8, also known as Tollo, and -9) have been found to play important roles in early development and the immune response [6][7][8][9][10][11][12]. Toll-2, along with Toll-1, plays a key role as an adhesion molecule in Drosophila compartment boundaries [13,14] and salivary gland morphogenesis [15]. Encoding more leucine-rich repeats (LRRs) is a shared feature that is believed to give Toll-2, -6, -7, and -8 rigid interaction and common roles in Drosophila embryonic elongation [16]. In addition to its role in development, 18-Wheeler is reported to have antibacterial activity in Drosophila larvae [17]. Previous studies have reported that Toll-5 induces the mRNA expression of the gene encoding for anti-microbial peptides (AMPs) such as drosomycin and metchnikowin [18]. Moreover, Toll-6 and Toll-7 participate in several functions within the Drosophila nervous system, including the wiring of neural circuitry, removal of apoptotic debris from neurons [16], motor axon targeting, neuronal survival [19], and axon and dendrite targeting [20]. Among the wide variety of biological . The phylogenetic tree was constructed using MEGA7 using the maximum likelihood method and 1000 bootstrap replicates (the numbers at the nodes indicate bootstrap support). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The phylogenetic tree was constructed based on amino acid sequence alignments using several protein sequences, including those of TcToll, Tribolium castaneum protein Toll (XP_967796.2); DmTollIsoformC, Drosophila melanogaster Toll isoform C (NP_001262995.1); AaToll, Aedes aegypti protein toll (XP_021708718.1); LsToll-likeX1, Lucilia sericata protein Toll-like isoform (X1 XP_037811182.1); BmTollX2, Bombyx mori protein Toll isoform X2 (XP_037870104.1); GmToll-like, Galleria mellonella protein Toll-like (XP_031767681.1); MsToll, Manduca sexta protein Toll (XP_037303038.1); NfToll, Nylanderia fulva protein Toll (XP_029178173.1); and HvTLR7, Homalodisca vitripennis Toll-like receptor 7 (XP_046670491.1). MmTLR2X1 Mus musculus Toll-like receptor 2 isoform X1 (XP_006501523.1) amino acid sequences were used as an outgroup.

Expression Analysis of TmToll-3 Transcripts
We next investigated the expression pattern of TmToll-3 by qRT-PCR at different developmental stages (egg, young-instar larvae, late-instar larvae, prepupae, 1-to 7-day-old pupae, and 1-to 5-day-old adults). Our results demonstrated that the TmToll-3 expression level was high in the embryonic stage but then gradually decreased between the pre-pupal and late-pupal stages (Figure 2A). We further examined the transcript levels of TmToll-3 in different tissues of late-instar larvae and adults. As shown in Figure 2B, TmToll-3 expression was relatively high in the integument of late-instar larvae and low in the fat bodies and Malpighian tubules. In contrast, in adult tissues, TmToll-3 was predominantly expressed in the gut and to a lesser extent in other tissues, including fat bodies, ovaries, and . The phylogenetic tree was constructed using MEGA7 using the maximum likelihood method and 1000 bootstrap replicates (the numbers at the nodes indicate bootstrap support). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The phylogenetic tree was constructed based on amino acid sequence alignments using several protein sequences, including those of TcToll, Tribolium castaneum protein Toll (XP_967796.2); DmTollIsoformC, Drosophila melanogaster Toll isoform C (NP_001262995.1); AaToll, Aedes aegypti protein toll (XP_021708718.1); LsToll-likeX1, Lucilia sericata protein Toll-like isoform (X1 XP_037811182.1); BmTollX2, Bombyx mori protein Toll isoform X2 (XP_037870104.1); GmToll-like, Galleria mellonella protein Toll-like (XP_031767681.1); MsToll, Manduca sexta protein Toll (XP_037303038.1); Nf Toll, Nylanderia fulva protein Toll (XP_029178173.1); and HvTLR7, Homalodisca vitripennis Toll-like receptor 7 (XP_046670491.1). MmTLR2X1 Mus musculus Toll-like receptor 2 isoform X1 (XP_006501523.1) amino acid sequences were used as an outgroup.

Expression Analysis of TmToll-3 Transcripts
We next investigated the expression pattern of TmToll-3 by qRT-PCR at different developmental stages (egg, young-instar larvae, late-instar larvae, prepupae, 1-to 7-day-old pupae, and 1-to 5-day-old adults). Our results demonstrated that the TmToll-3 expression level was high in the embryonic stage but then gradually decreased between the pre-pupal and late-pupal stages (Figure 2A). We further examined the transcript levels of TmToll-3 in different tissues of late-instar larvae and adults. As shown in Figure 2B, TmToll-3 expression was relatively high in the integument of late-instar larvae and low in the fat bodies and Malpighian tubules. In contrast, in adult tissues, TmToll-3 was predominantly expressed in the gut and to a lesser extent in other tissues, including fat bodies, ovaries, and testes ( Figure 2C). Unlike in the larvae, the lowest expression of TmToll-3 was detected in the integument of T. molitor adults. testes ( Figure 2C). Unlike in the larvae, the lowest expression of TmToll-3 was detected in the integument of T. molitor adults.

Figure 2.
Developmental stage-and tissue-specific expression patterns of TmToll-3 measured by qRT-PCR. (A) Relative mRNA expression levels of TmToll-3 in eggs, young larvae, late-instar larvae, pre-pupae, 1-to 7-day-old pupae (P1-P7), and 1-to 5-day-old adults (A1-A5). The expression levels were highest in the eggs. The mRNA expression decreased at the larval stage and was lowest at the young larval stage. TmToll-3 tissue expression patterns in late instar larvae (B) and adults (C) were also examined. Total RNA was extracted from different tissues, including the integument, Malpighian tubule (MT), gut (G), hemolymph (HL), and fat bodies (FB) of late instar larvae and the IT, MT, G, hemolymph, FB, ovary (OV), and testis (TE) of 5-day-old adults. Total RNA was isolated from 20 mealworms, and T. molitor 60S ribosomal protein 27a (TmL27a) primers were used as an internal control (n = 3). Comparisons between groups were made via one-way ANOVA and Tukey's multiple-range test. Different letters above each bar indicate statistically significant differences according to Tukey's multiple-range test (p < 0.05).
Next, to determine whether TmToll-3 expression was regulated in response to immune challenge, we further examined the temporal changes in TmToll-3 mRNA expression in T. molitor larvae after infection with either Gram-negative (E. coli), or Gram-positive (S. aureus) bacteria, or a fungus (C. albicans). In brief, total RNA was isolated from control and immune-challenged larvae (10th or 11th instar) at 3, 6, 9, 12, and 24 h post-infection, followed by reverse-transcription and qRT-PCR using TmToll-3-specific primers. As shown in Figure 3A-D, TmToll-3 was induced after challenging the larvae with S. aureus and C. albicans. However, in the case of E. coli infection, except for Malpighian tubules in which the TmToll-3 mRNA levels were marginally increased within 6 to 12 h post-infection, relevant expression in other tissues was insignificant compared with the PBS-injected larvae. Relative mRNA expression levels of TmToll-3 in eggs, young larvae, late-instar larvae, pre-pupae, 1-to 7-day-old pupae (P1-P7), and 1-to 5-day-old adults (A1-A5). The expression levels were highest in the eggs. The mRNA expression decreased at the larval stage and was lowest at the young larval stage. TmToll-3 tissue expression patterns in late instar larvae (B) and adults (C) were also examined. Total RNA was extracted from different tissues, including the integument, Malpighian tubule (MT), gut (G), hemolymph (HL), and fat bodies (FB) of late instar larvae and the IT, MT, G, hemolymph, FB, ovary (OV), and testis (TE) of 5-day-old adults. Total RNA was isolated from 20 mealworms, and T. molitor 60S ribosomal protein 27a (TmL27a) primers were used as an internal control (n = 3). Comparisons between groups were made via one-way ANOVA and Tukey's multiplerange test. Different letters above each bar indicate statistically significant differences according to Tukey's multiple-range test (p < 0.05).
Next, to determine whether TmToll-3 expression was regulated in response to immune challenge, we further examined the temporal changes in TmToll-3 mRNA expression in T. molitor larvae after infection with either Gram-negative (E. coli), or Gram-positive (S. aureus) bacteria, or a fungus (C. albicans). In brief, total RNA was isolated from control and immune-challenged larvae (10th or 11th instar) at 3, 6, 9, 12, and 24 h post-infection, followed by reverse-transcription and qRT-PCR using TmToll-3specific primers. As shown in Figure 3A-D, TmToll-3 was induced after challenging the larvae with S. aureus and C. albicans. However, in the case of E. coli infection, except for Malpighian tubules in which the TmToll-3 mRNA levels were marginally increased within 6 to 12 h post-infection, relevant expression in other tissues was insignificant compared with the PBS-injected larvae.  (10 6 cells/µL), and C. albicans (5 × 10 4 cells/µL). TmToll-3 mRNA expression was upregulated in response to all infectious sources and exhibited tissue-and time-dependent variations. The highest TmToll-3 expression level was observed in the Malpighian tubules in response to C. albicans challenge. PBS was used as an injection control and T. molitor 60S ribosomal protein 27a (TmL27a) primers were used as an internal control to quantify relative gene expression (n = 3). The asterisks indicate significant differences between infected and PBS-injected larval groups as determined using Student's ttest (p < 0.05). The vertical bars indicate means ± SD for each experimental condition (n = 20).

T. molitor Larval Mortality Assay
Given that our findings indicated that TmToll-3 expression was induced after infection with S. aureus, C. albicans, and E. coli, we next sought to determine whether TmToll-3 played a role in the immune response against bacteria and fungi by monitoring the survival rates of infected T. molitor larvae after treatment with either control dsRNA (TmVer) or TmToll-3 dsRNA. As illustrated in Figure 4A, TmToll-3 mRNA levels decreased by 75% in larvae 7 days after injection with TmToll-3 dsRNA compared with those treated with control dsRNA. After confirming the efficient knockdown of TmToll-3, we then challenged the dsTmToll-3-treated and control larvae by injecting them with 1 µL of bacterial (E. coli or S. aureus, 1 × 10 6 /µL) or fungal suspension (C. albicans, 5 × 10 4 /µL) and monitored their survival for 10 days. Surprisingly, the dsTmToll-3 larvae were significantly more susceptible to E. coli infection (36 vs. 13% mortality) compared with the dsTmVer (control) larvae, ( Figure 4B), whereas their survival rates after infection with S. aureus or C. albicans were not affected compared with the controls ( Figure 4C,D). PBS was used as an injection control and T. molitor 60S ribosomal protein 27a (TmL27a) primers were used as an internal control to quantify relative gene expression (n = 3). The asterisks indicate significant differences between infected and PBS-injected larval groups as determined using Student's t-test (p < 0.05). The vertical bars indicate means ± SD for each experimental condition (n = 20).

T. molitor Larval Mortality Assay
Given that our findings indicated that TmToll-3 expression was induced after infection with S. aureus, C. albicans, and E. coli, we next sought to determine whether TmToll-3 played a role in the immune response against bacteria and fungi by monitoring the survival rates of infected T. molitor larvae after treatment with either control dsRNA (TmVer) or TmToll-3 dsRNA. As illustrated in Figure 4A, TmToll-3 mRNA levels decreased by 75% in larvae 7 days after injection with TmToll-3 dsRNA compared with those treated with control dsRNA. After confirming the efficient knockdown of TmToll-3, we then challenged the dsTmToll-3-treated and control larvae by injecting them with 1 µL of bacterial (E. coli or S. aureus, 1 × 10 6 /µL) or fungal suspension (C. albicans, 5 × 10 4 /µL) and monitored their survival for 10 days. Surprisingly, the dsTmToll-3 larvae were significantly more susceptible to E. coli infection (36 vs. 13% mortality) compared with the dsTmVer (control) larvae, ( Figure 4B), whereas their survival rates after infection with S. aureus or C. albicans were not affected compared with the controls (Figure 4C,D). . The larval survival rates at 10 days post-microbial injection were 60% after E. coli injection and 100% after S. aureus and C. albicans injection compared with the survival rates of the dsTmVer-injected control group. Data were reported as averages of three biologically independent replicates. The asterisks indicate significant differences between the dsTmToll-3-and dsTmVer-injected groups. Survival analysis was performed using Kaplan-Meier plots (log-rank chi-squared test; * p < 0.05).

Effects of TmToll-3 RNAi on the Expression of AMP and Other Downstream Signaling Genes in Response to Microorganism Infection
Considering the contrasting findings related to the induction of TmToll-3 and the survival of T. molitor larvae after dsTmToll-3 treatment, we next sought to determine whether TmToll-3 mediated any of the observed effects through AMP production. Therefore, TmToll-3 was once again knocked out and the expression levels of 15 different AMP genes were measured following challenge with E. coli, S. aureus, and C. albicans. The aim of this experiment was to identify AMPs that were significantly induced upon infection by E. coli but reversed by TmToll-3 knockdown. In turn, this would suggest that TmToll-3 at least partially mediates the activation of these AMPs in response to E. coli infection. Among the 15 AMPs tested, except for TmTenecin-2 and TmThaumatin like protein-1, the mRNA expression levels of all the AMPs were induced in response to E. coli infection, but pretreatment with TmToll-3 dsRNA suppressed their upregulation ( Figure 5A-O, Figure S2 and Files S1-S3). Interestingly, TmToll-3 knockdown also suppressed the mRNA levels of TmTenecin-1, TmTenecin-4, TmDefensin-like, TmCecropin, TmColeoptericin-A, TmAttacin-1a, TmAttacin-1b, and TmAttacin-2 in S. aureus-challenged larvae. Finally, none of the 15 AMPs showed significant responses to C. albicans infection, regardless of whether TmToll-3 was knocked down prior to infection.
To further examine the role of TmToll-3 in regulating the immune response against pathogens, we investigated how TmToll3 RNAi might affect the expression of Toll pathway-related transcription factor genes (TmDorsal1 and TmDorsal-2) and one Imd-related gene (TmRelish) using the specific primers listed in Table 1. Our results demonstrated that the expression of the Toll pathway-related genes was reduced by TmToll-3 RNAi (p < 0.05) following E. coli infection, whereas the level of TmRelish was upregulated ( Figure 6). This . The larval survival rates at 10 days post-microbial injection were 60% after E. coli injection and 100% after S. aureus and C. albicans injection compared with the survival rates of the dsTmVer-injected control group. Data were reported as averages of three biologically independent replicates. The asterisks indicate significant differences between the dsTmToll-3-and dsTmVer-injected groups. Survival analysis was performed using Kaplan-Meier plots (log-rank chi-squared test; * p < 0.05).

Effects of TmToll-3 RNAi on the Expression of AMP and Other Downstream Signaling Genes in Response to Microorganism Infection
Considering the contrasting findings related to the induction of TmToll-3 and the survival of T. molitor larvae after dsTmToll-3 treatment, we next sought to determine whether TmToll-3 mediated any of the observed effects through AMP production. Therefore, TmToll-3 was once again knocked out and the expression levels of 15 different AMP genes were measured following challenge with E. coli, S. aureus, and C. albicans. The aim of this experiment was to identify AMPs that were significantly induced upon infection by E. coli but reversed by TmToll-3 knockdown. In turn, this would suggest that TmToll-3 at least partially mediates the activation of these AMPs in response to E. coli infection. Among the 15 AMPs tested, except for TmTenecin-2 and TmThaumatin like protein-1, the mRNA expression levels of all the AMPs were induced in response to E. coli infection, but pretreatment with TmToll-3 dsRNA suppressed their upregulation ( Figure 5A-O, Figure S2 and Files S1-S3). Interestingly, TmToll-3 knockdown also suppressed the mRNA levels of TmTenecin-1, TmTenecin-4, TmDefensin-like, TmCecropin, TmColeoptericin-A, TmAttacin-1a, TmAttacin-1b, and TmAttacin-2 in S. aureus-challenged larvae. Finally, none of the 15 AMPs showed significant responses to C. albicans infection, regardless of whether TmToll-3 was knocked down prior to infection.
suggests that TmToll-3 can positively regulate genes downstream of the Toll signaling pathway and that TmToll-3 functions through the Toll signaling pathway to regulate AMP expression.  To further examine the role of TmToll-3 in regulating the immune response against pathogens, we investigated how TmToll3 RNAi might affect the expression of Toll pathwayrelated transcription factor genes (TmDorsal1 and TmDorsal-2) and one Imd-related gene (TmRelish) using the specific primers listed in Table 1. Our results demonstrated that the expression of the Toll pathway-related genes was reduced by TmToll-3 RNAi (p < 0.05) following E. coli infection, whereas the level of TmRelish was upregulated ( Figure 6). This suggests that TmToll-3 can positively regulate genes downstream of the Toll signaling pathway and that TmToll-3 functions through the Toll signaling pathway to regulate AMP expression. Table 1. Sequences of the primers used in this study.

Discussion
Our assessment of the expression of AMPs in T. molitor in response to TmToll-3 knockdown and pathogen challenge provides key insights into the mechanisms that govern insect innate immunity. Upstream of this response is the recognition of microbial particles by special pattern recognition receptors (PRRs) that trigger proteolytic cascades, leading to the activation of signaling pathways responsible for AMP production [41,42]. While exploring the immunological roles of TmToll-3 in T. molitor, we found that Toll receptors and their possible ligands, 9 Spzs isoforms, reduced the induction of several AMPs [31][32][33][34][35], which we found to be suppressed by TmToll-3 RNAi after E. coli challenge. A previous study reported that out of the 9570 putative orthologs of annotated T. castaneum genes, 213 were related to immune functions in infected T. molitor [43]. While determining which of these receptors were involved in AMP production, we demonstrated that TmToll2 and TmToll7 participated in the immune response by regulating specific NF-kB transcription factors through preliminary knockdown studies [30,37].
The dynamic expression pattern of Toll proteins has also been reported in Drosophila [44]. Our results indicated that the mRNA of TmToll-3 was not only expressed during the embryonic stage but also in 5-day-old pupae, suggesting that Tolls play important roles throughout the insect's lifespan. Furthermore, unlike other Tolls in non-infected T. molitor, the highest TmToll-3 expression levels were observed in the integument of larvae and the gut of adults in our tissue-specific gene expression experiments, suggesting that similar to the Drosophila, this protein is closely linked to cuticle formation during T. molitor metamorphosis [45]. It would thus be interesting to investigate the evolutionary and developmental crosstalk between chitin and Toll expression and distribution. Unlike other Tolls in T. molitor, the mRNA expression of TmToll-3 in the most important immune organs of larvae exhibited low induction upon pathogen infection. However, similar to the expression of TmToll-2 [30], the main response occurred following C. albicans injection.
Therefore, for the remainder of our study, we focused on the effects of TmToll3 silencing in larvae. Unlike in Drosophila, where E. coli DAP-type peptidoglycans can only activate Imd signaling, purified PGRP-SA and GNBP1 (upstream of Toll receptors) mediate the cleavage of pro-Spätzle in T. molitor larvae, thus demonstrating that E. coli is able to activate Toll signaling in this insect [39,46]. Similarly, both PGN and LPS can induce the expression of AMP genes in Lepidoptera such as Bombyx mori and Manduca sexta [47]. Moreover, Drosophila might also be more sensitive to PGN than to LPS because it carries 13 genes that belong to the PGRP family and therefore expresses a wider repertoire of PGRP proteins [48]. Consistent with previous findings, the decreased survival rates of TmToll-3-silenced T. molitor larvae following E. coli infection observed herein suggested that humoral innate immunity could occur via TmToll-3. TmToll3 silencing in larvae rendered larvae more susceptible to E. coli infection and suppressed certain AMP genes induced by E. coli challenge, including TmTenesin-1, -4, TmDefensin, TmDefensin-like, TmColeoptericin-A, -B, -C, and TmAttacin-1a, -1b, -2, all four of which belong to AMP families known to exhibit antibacterial activity against Gram-negative bacteria [49][50][51][52]. TmToll-7 and TmToll-2 have been reported to activate their downstream NF-kB transcription factor, which leads to the induction of immune response genes including AMP genes [30,37]. Other studies have reported orthologs of transcription factors in T. molitor, including two Dif orthologs and two Relish orthologs [43]. Additionally, recent studies have characterized the signal activation further upstream of T. molitor NF-kB transcription factors and their relevant final effectors following bacterial and fungal infection [53,54]. Based on our findings that TmToll-3 RNAi inhibits AMP expression, we hypothesize that TmToll-3 in T. molitor plays an important role in mediating nuclear translocation of transcription factors for activating AMP gene expression. Our findings demonstrated that, after TmToll-3 activation in T. molitor, Dif, TmDorx2, and potentially a Dif-Relish heterodimer stimulate the production of AMP genes as the final effectors. Our findings also demonstrated the lack of AMP specificity after the activation of NF-kB transcription factors following infection with different pathogens.
Additional studies are thus needed to shed light on what makes TmToll-3 different from other Tolls in T. molitor that mediate immune responses, as well as whether their distinctions and similarities stemmed from evolutionary convergence or another mechanism entirely. Additionally, it would be interesting to assess which of the nine identified Spz ligands interact directly with TmToll3 (or other elements) and whether their interactions are relevant to immunity and/or development in T. molitor.

Insect Rearing and Microbial Infection
T. molitor larvae were reared on a wheat bran diet at 27 ± 1 • C, a 60 ± 5% relative humidity, and under dark conditions. All experiments were conducted with 10-12th instar larvae. To investigate the immunological function of TmToll-3 against infections, three microorganisms, including E. coli K12, Staphylococcus aureus RN4220, and Candida albicans were used. Overnight cultures of E. coli, S. aureus, and C. albicans were grown in Luria-Bertani (LB) broth and Sabouraud Dextrose broth at 37 • C, respectively. The microorganisms were harvested, washed, and suspended in phosphate-buffered saline (PBS, pH 7.0) by centrifugation at 3500 rpm for 10 min, and the concentrations were measured at OD600. Finally, 10 6 cells/µL of E. coli and S. aureus and 5 × 10 4 cells/µL of C. albicans were injected into the larvae.
RNA was isolated using the Clear-S Total RNA Extraction Kit (Invirustech Co., Gwangju, Republic of Korea) according to the manufacturer's instructions. Next, 2 µg of total RNA was used as a template to synthesize cDNA via Oligo(dT)-primed synthesis [12][13][14][15][16][17][18] under the following reaction conditions: 72 • C for 5 min, 42 • C for 1 h, and 94 • C for 5 min. These procedures were conducted using a MyGenie96 Thermal Block (Bioneer, Daejeon, Korea) and AccuPower ® RT PreMix (Bioneer Co., Daejeon, South Korea) according to the manufacturer's instructions. The cDNA was then stored at -20 • C until further use. To investigate the expression levels of TmToll-3 transcripts, qRT-PCR was conducted using AccuPower ® 2X GreenStar qPCR Master Mix (Bioneer), using the synthesized cDNAs as templates and gene-specific primers (TmToll-3_qPCR_Fw and TmToll-3_qPCR_Rv) ( Table 1) at an initial denaturation step at 94 • C for 2 min followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 53 • C for 30 s, and extension at 72 • C for 30 s, with a final extension step at 72 • C for 5 min. T. molitor ribosomal protein (TmL27a) was used as an internal control and relative gene expression was calculated via the 2 −∆∆Ct method.

RNA Interference Analysis
The PCR product (510 bp sequence) containing the T7 promoter sequences was amplified using the AccuPower ® Pfu PCR PreMix polymerase with TmToll-3_T7_Fw and Rv primers (Table 1) using the same PCR conditions described above. dsRNA for TmToll-3 was synthesized using the AmpliScribe T7-Flash Transcription Kit (Epicentre, Madison, WI, USA) and was purified with PCI (Phenol:Chloroform:Isopropyl alcohol mixture), followed by ammonium acetate purification and ethanol precipitation. Finally, 2 µg of synthesized dsTmToll-3 was injected into 10-11th instar larvae for gene silencing and T. molitor vermilion (TmVer) double-strand RNA (dsTmVer) was used as a control [57].

Effect of TmToll-3 Knockdown on the Response to Microorganisms
To investigate the effect of TmToll-3 knockdown on the systemic response to microbial infection, 10 6 cells/µL of E. coli and S. aureus, and 5 × 10 4 cells/µL of C. albicans were injected into dsTmToll-3-treated T. molitor larvae, respectively. Larval mortality was monitored up to 10 days post-injection of microorganisms. Ten insects per group were used for this assay and the experiments were replicated three times.

Effect of TmToll-3 RNAi on AMP Expression in Response to Microorganisms
To characterize the function of TmToll-3 on the humoral innate immune response, gene knockdown experiments were conducted through TmToll-3 RNAi injection, after which the T. molitor larvae were infected with the microorganisms (E. coli, S. aureus, and C. albicans) via injection. Samples were then collected at 24 h post-infection. PBS was used as an injection control and dsTmVer-treated T. molitor was used as a negative control. qRT-PCR was then conducted to characterize the temporal expression patterns of 15 antimicrobial peptide (AMP) genes, including TmTene-1 ( Figure 5A Figure 5N: TmThaumatin-like protein-1), and TmTLP-2 ( Figure 5O: TmThaumatin-like protein-2). Table 1 summarizes the sequences of the gene-specific primers used in this study.

Statistical Analysis
All experiments were performed in triplicate and all data are presented as means ± standard error (SE). Differences between groups were evaluated via one-way analysis of variance (ANOVA) and Tukey's multiple range tests. p-values < 0.05 were considered statistically significant.  Institutional Review Board Statement: Not applicable.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article Supplementary Materials.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.